Precise Spatiotemporal Control Research Articles

Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice.

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk. As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (µFIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The γ-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control. As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Azobenzene‐Based Photoswitches Facilitating Reversible Regulation of Kinesin and Myosin Motor Systems for Nanotechnological Applications

AbstractThe past decade has witnessed several significant efforts made towards implementing the biomolecular functions of motor proteins for nanotechnological applications. However, employing motor protein systems in artificial environments demands for a precise and flexible spatiotemporal control mechanism. Light being an ideal external stimulus offers major advantages, allowing better spatial and temporal resolution compared to other diffusion‐based approaches. Robust photochromic properties of azobenzene make it a prime candidate that could be incorporated into biologically active molecules to realize two‐way regulation of motor protein function. In this Minireview, we summarize the design strategies of azobenzene‐based photoswitches for reversibly regulating kinesin‐ and myosin‐driven shuttle movements and briefly discuss photoisomerization‐induced modulation in motor protein activity.

Biochemical Stimulation of Immune Cells and Measurement of Mechanical Responses Using Atomic Force Microscopy.

This manuscript details methods to ligate cell-surface receptors on live cells with precise spatiotemporal control using an atomic force microscope (AFM) to deliver ligands. This approach can be used to image cellular responses upon activating T cell receptors when the AFM is mounted on an optical microscope. Moreover, the AFM measures forces generated by the cell during the contact. Using AFM to trigger cellular responses adds an important capability to the field of mechanobiology. We describe how to incorporate anti-CD3 antibodies or other molecules onto an AFM cantilever and how to use AFM to activate T cells. © 2019 by John Wiley & Sons, Inc.

A "Clickable" Photoconvertible Small Fluorescent Molecule as a Minimalist Probe for Tracking Individual Biomolecule Complexes.

Photoconvertible fluorophores can enable the visualization and tracking of a specific biomolecules, complexes, and cellular compartments with precise spatiotemporal control. The field of photoconvertible probes is dominated by fluorescent protein variants, which can introduce perturbations to the target biomolecules due to their large size. Here, we present a photoconvertible small molecule, termed CPX, that can be conjugated to any target through azide-alkyne cycloaddition ("click" reaction). To demonstrate its utility, we have applied CPX to study (1) trafficking of biologically relevant synthetic vesicles and (2) intracellular processes involved in transmission of α-synuclein (αS) pathology. Our results demonstrate that CPX can serve as a minimally perturbing probe for tracking the dynamics of biomolecules.

Microfluidics tubing as a synthesizer for ordered microgel networks.

Ordered microgel networks have undergone extensive research and shown translational promise in tissue engineering, precision and regenerative medicine, controlled delivery, optics and electronics, etc. Here, we introduce a new low-cost and efficient synthesizer for ordered microgel networks. The gel precursor microdroplets are formulated and incubated in a microfluidics tubing system to obtain tailorable and reproducible microgels, which are then patterned into networks under the precise spatiotemporal control of the tubing system and integrated either by crosslinking the microgel interfaces or by forming lipid bilayers at the interfaces. The system can synthesize ordered networks out of heterogeneous microgels by withdrawing multi-phase cell-laden or acellular gel precursors into the tubing and gelation, or out of homogeneous microgels by simultaneously injecting gel precursors and immiscible oil into the tubing and gelation. The ordered gel networks are synthesized at the tubing outlet or within a piece of enlarging tubing, where the microgels are collided and glued in defined sequences.

Fluorogenic "Photoclick" Labeling and Imaging of DNA with Coumarin-Fused Tetrazole in Vivo.

Photoclickable fluorogenic probes will enable visualization of specific biomolecules with precise spatiotemporal control in their native environment. However, the fluorogenic tagging of DNA with current photocontrolled clickable probes is still challenging. Herein, we demonstrated the fast (19.5 ± 2.5 M-1 s-1) fluorogenic labeling and imaging of DNA in vitro and in vivo with rationally designed coumarin-fused tetrazoles under UV LED photoirradiation. With a water-soluble, nuclear-specific coumarin-fused tetrazole (CTz-SO3), the metabolically synthesized DNA in cultured cells was effectively labeled and visualized, without fixation, via "photoclick" reaction. Moreover, the photoclickable CTz-SO3 enabled real-time, spatially controlled imaging of DNA in live zebrafish.

Photoswitchable Antimetabolite for Targeted Photoactivated Chemotherapy.

The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects. Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human dihydrofolate reductase (DHFR), as a photochromic analogue of methotrexate, a widely prescribed chemotherapeutic drug to treat cancer and psoriasis. Quantification of the light-regulated DHFR enzymatic activity, cell proliferation, and in vivo effects in zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration and that it is nearly inactive in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small molecules and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse effects.

Ultrasound-Responsive Nanoparticulate for Selective Amplification of Chemotherapeutic Potency for Ablation of Solid Tumors.

Precision medicine requests preferential transportation of the pharmaceutical substances to the pathological site and impartation of localized therapeutic activities to the targeted cells. To accomplish this goal, we attempted a facile nanoscaled ultrasound-responsive delivery system, characterized by doxorubicin assembled with an amphiphilic copolymer (multiple of hydrophobic stearic segments tethered onto the hydrophilic pullulan backbone through ultrasound-labile oxyl-alkylhydroxylamine linkage). As a consequence of the strategically installed ultrasound-labile oxyl-alkylhydroxylamine linkage to elicit the tailored segregation of the hydrophilic pullulan and the hydrophobic stearic segments upon ultrasound impetus, the constructed nanoscaled self-assembly presented distinctive structural destabilization behaviors and afforded spatiotemporal controlled liberation of the cytotoxic drugs. It is worthy to note that the ultrasound was determined to markedly lower the IC50 of the proposed system from over 10 μg/mL to 2.33 μg/mL (approximate 4-fold), thereby serving as a facile impetus to amplify the cytotoxic potency of the proposed drug delivery vehicles. Furthermore, drastic tumor ablation was validated by dosage of the proposed doxorubicin delivery system to T41 tumor-bearing mice accompanied by the tumor-localized ultrasound impetus, while no observable adverse side effect was confirmed. Therefore, the results advocated our ultrasound-responsive delivery vehicle as a tempting strategy for precise spatiotemporal control of the release of the drug cargo, thus affording selectively amplified cytotoxic potency to the ultrasound-imposed site, which should be highlighted as important progress toward precision medicine.

Protein Engineering Strategies to Expand CRISPR-Cas9 Applications.

The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study and engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene networks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided site-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target. CRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene therapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a precise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a protospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for CRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise spatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment and in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9 and generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for expanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or develop novel protein functions through domain fusion or splitting, rational design, and directed evolution.

Photocontrol of Drug Release from Supramolecular Hydrogels with Green Light.

Photoresponsive smart materials transform light energy into sophisticated functions. They find increasing biomedical applications in light-induced drug-release and photopharmacology, because they can provide the desired therapeutic effect locally due to precise spatiotemporal dosage control. However, the majority of reported studies rely on cytotoxic UV light that penetrates tissues poorly. Here, we report the first drug-releasing system based on photochromic low molecular weight supramolecular hydrogels that is triggered with visible light. We demonstrated green-light-induced release of structurally unmodified antibiotic, anticancer, and anti-inflammatory drugs under physiological conditions. Using the antibiotic-loaded gel, we selectively inhibited bacterial growth with green light.

Photo-Controlled Release of Small Signaling Molecules to Induce Biological Responses.

Chemical modifications of proteins or cofactors, including acetylation and oxidation of amino acid residues of various signal proteins, whether transient or successive, play key roles in modulating biological functions. Small molecules that have signaling functions in biological systems through the chemical modification of proteins include nitric oxide (NO), hydrogen peroxide, carbon monoxide, and hydrogen sulfide. To investigate the pathophysiological roles of these molecules, caged compounds have been developed that allow precise spatiotemporal control of the release of these species in response to photoirradiation in the ultraviolet or visible region. For example, photocontrollable NO releasers can regulate the responses of blood vessels in vivo and ex vivo. In addition, photocontrollable (caged) inhibitors of histone deacetylase (HDAC) can be used to regulate HDAC activity in response to photoirradiation. Such photocontrol technology has provided chemical tools for a variety of biological studies, including investigations of epigenetic mechanisms.

Structural basis for the regulation of inositol trisphosphate receptors by Ca2+ and IP3.

Inositol trisphosphate receptors (IP3R) are ubiquitous Ca2+-permeable channels that mediate release of Ca2+ from the endoplasmic reticulum to regulate numerous processes including cell division, cell death, differentiation and fertilization. IP3R is activated by both IP3 and its permeant ion Ca2+. At high concentrations, however, Ca2+ inhibits activity ensuring precise spatiotemporal control over intracellular Ca2+. Despite extensive characterization of IP3R, the mechanisms by which these molecules control channel gating have remained elusive. Here, we present structures of full-length human type 3 IP3R in ligand-bound and ligand-free states. Multiple IP3-bound structures demonstrate that the large cytoplasmic domain provides a platform for propagation of long-range conformational changes to the ion conduction gate. Structures in the presence of Ca2+ reveal two Ca2+ binding sites that induce the disruption of numerous interactions between subunits, thereby inhibiting IP3R. These structures thus begin to provide a mechanistic basis for understanding the regulation of IP3R.

Host-guest self-assembly toward reversible visible-light-responsive switching for bacterial adhesion

Here we report a facile method to construct reversible visible-light-responsive switching from antibacterial to bioadhesion by host-guest self-assembly of β-cyclodextrin (β-CD) and azobenzene functionalized polycation/polyanion. The visible-light-responsible azobenzene functionalized polycation, poly{6-[(2,6-dimethoxyphenyl)azo-4-(2′,6′-dimethoxy)phenoxy]propyl dimethylaminoethyl methacrylate-random-poly(2-(N,N-dimethylaminoethyl) methacrylate) (Azo-PDMAEMA), was synthesized via quaternization reaction between 2,6,2′,6′-tetramethoxy-4-(3-bromopropoxy)azobenzene (AzoOMeBr) and poly(2-(N,N-dimethylaminoethyl) methacrylate) (PDMAEMA), and the polyanion, poly{6-[(2,6-dimethoxyphenyl)azo-4-(2′,6′-dimethoxy) phenoxy]hexyl acrylate-random-acrylic acid} (Azo-PAA), was synthesized via esterification reaction between 2,6,2′,6′-tetramethoxy-4-(6-hydroxyhexyloxy) azobenzene (AzoOMeOH) and poly(acryloyl chloride) (PAC) and subsequent hydrolysis reactions. The switch surface could be achieved via the alternate host-guest assembly of Azo-PDMAEMA and Azo-PAA onto a β-CD-terminated substratum (Sub-CD) through visible light irradiation. The positively charged Azo-PDMAEMA with quaternary ammonium groups exhibited antimicrobial properties and few bacteria were adhered on the surface, while the negatively charged Azo-PAA with carboxyl acid groups exhibited excellent bioadhesive properties and a large number of bacteria were adhered. Interestingly, the switch between antibacterial and bioadhesive could be realized upon visible light irradiation via alternate assembly of Azo-PDMAEMA and Azo-PAA. The proposed approach to manufacturing visible-light-responsive surface with reversible and alterable biofunctionality switching between antibacterial and bioadhesive is simple and efficient, which is promising for preparation of multifunctional polymeric surfaces to encounter multifarious demands for the biomedical and biotechnological applications. Statement of SignificanceLight has attracted great attention in building biointerfaces for its precise spatiotemporal control and convenient operation. However, UV light may damage to biological samples and living tissues, which will limit its applications. This study demonstrates a novel visible-light-responsive surface fabricated through reversible assembly of azobenzene functionalized polycations/polyanions on cyclodextrin (CD)-terminated substrate by host-guest interactions between the visible-light-responsive azobenzene mAzo and CD, which has not been examined previously. It is noted that the azobenzene functionalized polycations show strong antibacterial activities, while the polyanions show excellent bioadhesive properties, as can be switched through the alternate assembly upon visible-light irradiation. This facile and versatile approach to visible-light-responsive surfaces holds great potential for switching of bioadhesion.

Synthetic far-red light-mediated CRISPR-dCas9 device for inducing functional neuronal differentiation

The ability to control the activity of CRISPR-dCas9 with precise spatiotemporal resolution will enable tight genome regulation of user-defined endogenous genes for studying the dynamics of transcriptional regulation. Optogenetic devices with minimal phototoxicity and the capacity for deep tissue penetration are extremely useful for precise spatiotemporal control of cellular behavior and for future clinic translational research. Therefore, capitalizing on synthetic biology and optogenetic design principles, we engineered a far-red light (FRL)-activated CRISPR-dCas9 effector (FACE) device that induces transcription of exogenous or endogenous genes in the presence of FRL stimulation. This versatile system provides a robust and convenient method for precise spatiotemporal control of endogenous gene expression and also has been demonstrated to mediate targeted epigenetic modulation, which can be utilized to efficiently promote differentiation of induced pluripotent stem cells into functional neurons by up-regulating a single neural transcription factor, NEUROG2 This FACE system might facilitate genetic/epigenetic reprogramming in basic biological research and regenerative medicine for future biomedical applications.

Design and development of a robust photo-responsive block copolymer framework for tunable nucleic acid delivery and efficient gene silencing

Nucleic acid delivery offers tremendous potential for the treatment of acquired and hereditary diseases. Despite limited successes, the use of nucleic acid therapies has been hampered by the lack of safe and efficient delivery approaches. To address this challenge, Epps, Sullivan, and coworkers developed a new nucleic acid delivery framework predicated on a photo-responsive cationic block copolymer (BCP) that enabled tunable nucleic acid binding and precise spatiotemporal control over gene expression. This innovative platform, in which the polymer moieties directly responsible for nucleic acid complexation could be cleaved from the polymer upon photo-stimulation, significantly enhancing nucleic acid release. Furthermore, temporal control over polyplex disassembly facilitated the development of a simple, and potentially universal, kinetic modeling scheme for intracellular small interfering RNA (siRNA), messenger RNA, and protein concentrations, and that model was quantitatively validated using various genes across several animal cell lines and human primary cells. This versatile BCP-based framework easily accommodated: anionic excipients that increased siRNA potency by ~200% (on a per mass basis) over comparable polyplex systems; quantum dots that unlocked theranostic applications without impacting silencing performance; and small-molecule lipid co-formulations that enhanced transfection in human primary cells. Altogether, the system described herein shows great promise for the clinical translation of gene therapeutics. A new nucleic acid delivery framework, predicated on a photo-responsive cationic block copolymer (BCP), was used to precisely tune nucleic acid binding and provide spatiotemporal control over gene expression. This innovative platform leveraged a macromolecular design in which the polymer moieties directly responsible for nucleic acid complexation were cleaved from the polymer upon photo-stimulation, significantly enhancing nucleic acid release. Temporal control over polyplex disassembly facilitated development of a potentially universal, kinetic modeling scheme for gene silencing. Furthermore, this versatile BCP-based framework easily accommodated anionic excipients and quantum dot imaging constructs.

Ru(II) Polypyridyl Complexes Derived from Tetradentate Ancillary Ligands for Effective Photocaging.

Metal complexes have many proven applications in the caging and photochemical release of biologically active compounds. Photocaging groups derived from Ru(II) traditionally have been composed of ancillary ligands that are planar and bi- or tridentate, such as 2,2'-bipyridine (bpy), 2,2':6',2″-terpyridine (tpy), and 1,10-phenanthroline (phen). Complexes bearing ancillary ligands with denticities higher than three represent a new class of Ru(II)-based photocaging groups that are grossly underdeveloped. Because high-denticity ancillary ligands provide the ability to increase the structural rigidity and control the stereochemistry, our groups initiated a research program to explore the applications of such ligands in Ru(II)-based photocaging. Ru(TPA), bearing the tetradentate ancillary ligand tris(2-pyridylmethyl)amine (TPA), has been successfully utilized to effectively cage nitriles and aromatic heterocycles. Nitriles and aromatic heterocycles caged by the Ru(TPA) group show excellent stability in aqueous solutions in the dark, and the complexes can selectively release the caged molecules upon irradiation with light. Ru(TPA) is applicable as a photochemical agent to offer precise spatiotemporal control over biological activity without undesired toxicity. In addition, Ru(II) polypyridyl complexes with desired photochemical properties can be synthesized and identified by solid-phase synthesis, and the resulting complexes show properties to similar to those of complexes obtained by solution-phase synthesis. Density functional theory (DFT) calculations reveal that orbital mixing between the π* orbitals of the ancillary ligand and the Ru-N dσ* orbital is essential for ligand photodissociation in these complexes. Furthermore, the introduction of steric bulk enhances the photoliability of the caged molecules, validating that steric effects can largely influence the quantum efficiency of photoinduced ligand exchange in Ru(II) polypyridyl complexes. Recently, two new photocaging groups, Ru(cyTPA) and Ru(1-isocyTPQA), have been designed and synthesized for caging of nitriles and aromatic heterocycles, and these complexes exhibit unique photochemical properties distinct from those derived from Ru(TPA). Notably, the unusually greater quantum efficiency for the ligand exchange in [Ru(1-isocyTPQA)(MeCN)2](PF6)2, Φ400 = 0.033(3), uncovers a trans-type effect in the triplet metal-to-ligand charge transfer (3MLCT) state that enhances photoinduced ligand exchange in a new manner. DFT calculations and ultrafast transient spectroscopy reveal that the lowest-energy triplet state in [Ru(1-isocyTPQA)(MeCN)2](PF6)2 is a highly mixed 3MLCT/3ππ* excited state rather than a triplet metal-centered ligand-field (3LF) excited state; the latter is generally accepted for ligand photodissociation. In addition, Mulliken spin density calculations indicate that a majority of the spin density in [Ru(1-isocyTPQA)(MeCN)2](PF6)2 is localized on the isoquinoline arm, which is opposite to the cis MeCN, rather than on the ruthenium center. This significantly weakens the Ru-N6 ( cis MeCN) bond, which then promotes the ligand photodissociation. This newly discovered effect gives a clearer perception of the interplay between the 3MLCT and 3LF excited states of Ru(II) polypyridyl complexes, which may be useful in the design and applications of ruthenium complexes in the areas of photoactivated drug delivery and photosensitizers.

Mediator subunit MED31 is required for radial patterning of Arabidopsis roots

Stem cell specification in multicellular organisms relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, in which the evolutionarily conserved Mediator coactivator complex plays an essential role. In Arabidopsis thaliana, SHORTROOT (SHR) and SCARECROW (SCR) orchestrate a transcriptional program that determines the fate and asymmetrical divisions of stem cells generating the root ground tissue. The mechanism by which SHR/SCR relays context-specific regulatory signals to the Pol II general transcription machinery is unknown. Here, we report the role of Mediator in controlling the spatiotemporal transcriptional output of SHR/SCR during asymmetrical division of stem cells and ground tissue patterning. The Mediator subunit MED31 interacted with SCR but not SHR. Reduction of MED31 disrupted the spatiotemporal activation of CYCLIND6;1 (CYCD6;1), leading to defective asymmetrical division of stem cells generating ground tissue. MED31 was recruited to the promoter of CYCD6;1 in an SCR-dependent manner. MED31 was involved in the formation of a dynamic MED31/SCR/SHR ternary complex through the interface protein SCR. We demonstrate that the relative protein abundance of MED31 and SHR in different cell types regulates the dynamic formation of the ternary complex, which provides a tunable switch to strictly control the spatiotemporal transcriptional output. This study provides valuable clues to understand the mechanism by which master transcriptional regulators control organ patterning.

Targeting Photoinduced DNA Destruction by Ru(II) Tetraazaphenanthrene in Live Cells by Signal Peptide.

Exploiting NF-κB transcription factor peptide conjugation, a Ru(II)-bis-tap complex (tap = 1,4,5,8-tetraazaphenanthrene) was targeted specifically to the nuclei of live HeLa and CHO cells for the first time. DNA binding of the complex within the nucleus of live cells was evident from gradual extinction of the metal complex luminescence after it had crossed the nuclear envelope, attributed to guanine quenching of the ruthenium emission via photoinduced electron transfer. Resonance Raman imaging confirmed that the complex remained in the nucleus after emission is extinguished. In the dark and under imaging conditions the cells remain viable, but efficient cellular destruction was induced with precise spatiotemporal control by applying higher irradiation intensities to selected cells. Solution studies indicate that the peptide conjugated complex associates strongly with calf thymus DNA ex-cellulo and gel electrophoresis confirmed that the peptide conjugate is capable of singlet oxygen independent photodamage to plasmid DNA. This indicates that the observed efficient cellular destruction likely operates via direct DNA oxidation by photoinduced electron transfer between guanine and the precision targeted Ru(II)-tap probe. The discrete targeting of polyazaaromatic complexes to the cell nucleus and confirmation that they are photocytotoxic after nuclear delivery is an important step toward their application in cellular phototherapy.

Biopolymers for Antitumor Implantable Drug Delivery Systems: Recent Advances and Future Outlook.

In spite of remarkable improvements in cancer treatments and survivorship, cancer still remains as one of the major causes of death worldwide. Although current standards of care provide encouraging results, they still cause severe systemic toxicity and also fail in preventing recurrence of the disease. In order to address these issues, biomaterial-based implantable drug delivery systems (DDSs) have emerged as promising therapeutic platforms, which allow local administration of drugs directly to the tumor site. Owing to the unique properties of biopolymers, they have been used in a variety of ways to institute biodegradable implantable DDSs that exert precise spatiotemporal control over the release of therapeutic drug. Here, the most recent advances in biopolymer-based DDSs for suppressing tumor growth and preventing tumor recurrence are reviewed. Novel emerging biopolymers as well as cutting-edge polymeric microdevices deployed as implantable antitumor DDSs are discussed. Finally, a review of a new therapeutic modality within the field, which is based on implantable biopolymeric DDSs, is given.

Enhancers: bridging the gap between gene control and human disease.

Enhancers are a class of regulatory elements essential for precise spatio-temporal control of gene expression during development and in terminally differentiated cells. This review highlights signature features of enhancer elements as well as new advances that provide mechanistic insights into enhancer-mediated gene control in the context of three-dimensional chromatin. We detail the various ways in which non-coding mutations can instigate aberrant gene control and cause a variety of Mendelian disorders, common diseases and cancer.